Synechococcus Protein Expression

The cyanobacterium Synechococcus elongatus is an excellent system for studying photosynthesis, circadian rhythms, nutrient regulation, environmental response, and lipid metabolism. Cyanobacteria, also known as blue-green algae, are the basis for a common platform for bioproduction of alcohols and chemicals, owing to their small genomes and the ease with which they can be genetically manipulated by natural transformation.

Better expression and a choice of purification tags

The multiple cloning site (MCS) in the GeneArt Synechococcus Protein Expression Vector is designed to provide constitutive and robust expression in S. elongatus. Expression is controlled by the psbA1 promoter, a ribosome binding site (RBS), and optional initiation and stop codons depending on where you clone your gene. Features of the vector include:

Targeted integration of your gene of interest into the S. elongatus genome

N-terminal 6His TEV and C-terminal V5-His epitope tags

Spectinomycin resistance gene for selection in Synechococcus

Flexible multiple-cloning site vector

The gene for β-glucuronidase (GUS) was cloned into vector pSyn_6 and transformed into S. elongatus (negative for GUS activity) following the manufacturer's recommended protocols. GUS activity produced using the pSyn_6 vector was more than 100-fold greater than previous observations using pSyn_1.

The gene for β-glucuronidase (GUS) was cloned into vector pSyn_6 and transformed into S. elongatus(negative for GUS activity) following the manufacturer's recommended protocols. GUS expression was measured by western blot analysis, which showed GUS protein representing >20% of total soluble protein. TSP = total soluble protein.

Greater than 80% integration efficiency

The transformation of S. elongatus PCC 7942 relies on homologous recombination between the cell’s chromosome and exogenous DNA that is not autonomously replicating and contains sequences homologous to the chromosome. The location of integration into the chromosome (neutral site, NS1) was developed as a cloning locus (Clerico et al., 2007) as it can be disrupted without any aberrant phenotype, thus allowing recombination of ectopic sequences. When transformed with vectors containing an antibiotic resistance cassette and neutral site sequences, a double homologous recombination event occurs between the neutral site vector and the S. elongatus chromosome. The selective marker (spectinomycin resistance) and the gene of interest driven by a promoter are inserted into the neutral site and the vector backbone (pUC) is lost, allowing the expression of recombinant genes in S. elongatus PCC 7942.

Gibco BG-11 Media—optimized for cyanobacteria

Gibco™ BG-11 Medium, oﬀered separately, is optimized for the growth and maintenance of select cyanobacteria including S. elongatus. The 1X formulation lets you avoid laborious media preparation steps. The bottle design makes manipulation in the biosafety cabinet easier, minimizes the risk of contamination, and helps you perform cell culture more consistently. Superior packaging and quality, greater reliability, and improved consistency in cyanobacterial culture result in better overall eﬃciency and more robust data.